Food additive to combat infectious diseases caused by marine bacterial pathogens

Abstract

The invention relates to a food additive for combating infectious diseases caused by marine bacterial pathogens, such as Piscirickettsia salmonis, and reducing the use of antibiotics in the salmon farming industry.

Description

[0001] FOOD ADDITIVE TO COMBAT DISEASES

[0002] INFECTIOUS DISEASES CAUSED BY MARINE BACTERIAL PATHOGENS

[0003] DESCRIPTIVE MEMORANDUM

[0004] Technical Field

[0005] The present invention relates, in general terms, to a food additive to combat infectious diseases caused by marine bacterial pathogens, and to reduce the use of antibiotics in the salmon industry.

[0006] Background

[0007] Chile is one of the world's leading aquaculture producers (Lulijwa et al., 2020), primarily producing salmonids, which account for 71.7% of total aquaculture production (AQUA, 2021). In 2020, the salmon farming industry produced over 1 million tons, mainly exporting Atlantic salmon (72%) and, to a lesser extent, coho salmon (19%) and rainbow trout (9%), representing earnings of over US$4.4 billion (AQUA, 2021). However, the growth in production has led to an increase in infectious diseases caused by various pathogens (Mohapatra et al., 2013), with Piscirickettsia salmonis responsible for approximately 50% of mortality in Atlantic salmon and rainbow trout and 13% in coho salmon (SERNAPESCA, 2023). This bacterium has affected the salmon industry for more than 30 years, generating losses of over US$700 million per year (Maisey et al., 2017).

[0008] P. salmonis is the etiological agent of salmonid rickettsial syndrome (SRS). It is a Gram-negative, pleomorphic, but generally coccoid bacterium, with a variable size between 0.2 µm and 1.5 µm (Rozas & Enriquez, 2014), and is facultatively intracellular (Gomez et al., 2009; Yañez et al., 2012). P. salmonis is able to survive and replicate within replicative vacuoles in phagocytic cells such as macrophages and monocyte-like cells (V. Rojas et al., 2009), and is able to persist inside phagocytic cells by evading phagosome-lysosome fusion (Gomez et al., 2013) through activation of the type IV secretion system (Gomez et al., 2013). Furthermore, it is capable of generating various virulence factors (Levipan et al., 2020; Marshall et al., 2012), producing siderophores (Calquín et al., 2018), generating vesicles in the outer membrane (Oliver et al., 2016), and secreting exotoxins (ME Rojas et al., 2013). On the other hand, P.salmonis is able to modulate different biological processes in the host, including the cellular and humoral immune response (Álvarez et al., 2016; Rozas-Sem, Peña, & Maldonado, 2018; Rozas-Serri, Peña, Amagada, et al., 2018), favoring the conditions for its replication.

[0009] In Chile, the most commonly used treatments for controlling *P. salmonis* are vaccines and antibiotics. Currently, 32 vaccines against piscichetsiosis are available in Chile (C. Figueroa et al., 2021; Happold et al., 2020). However, despite the large number of vaccines available, they are not effective in protecting against *P. salmonis*, merely delaying the first outbreak (Jakob et al., 2014). Therefore, the use of antibiotics is necessary to control this pathogen (Miranda et al., 2018). Chile is one of the countries that uses the most antibiotics for salmonid production. In 2022, approximately 304 tons were used as a treatment against *P. salmonis* (Sernapesca, 2023). This has generated several problems, including environmental contamination and the emergence of resistant bacterial isolates (J. Figueroa et al., 2019; Miranda et al., 2018; Saavedra et al.(2017), which is why it is necessary to seek new alternatives to the use of antibiotics for the control of P. salmonis. Regarding the aforementioned problems and the need to find new alternatives and modes of action for treatments against P. salmonis, the use of natural compounds capable of reducing the intracellular replication of the bacteria without affecting its viability is proposed, thus decreasing its ability to develop resistance. In this search, two natural compounds, Silibinin and Quercetin, have been identified with the aforementioned capacity, and are also capable of providing protection in salmonids when challenged with P. salmonis.

[0010] In the state of the art, information can be found on the use of Silibinin, but not for its use against infection caused by P. salmonis.

[0011] The article “A Synergy of the Nutritional Additives Taurine and Silymarin in Salmon Farming: Evaluation with the CHSE-214 Cell Model,” by Olivares, Sánchez, et al., indicates that the administration of silymarin (a complex formed by four flavonoids, where approximately 50% of the mixture corresponds to silybin) as a feed additive is a common practice that improves development and growth and has a range of uses, from enhancing the development of freshwater fish to its use in the grow-out stage of marine species. Silymarin is used as an immunomodulator, protects against toxic agents, and is believed to regulate glial cell function. In cell models, the effects of silymarin have been described as protective, suggesting an antioxidant role. This document does not mention its use in combating infectious diseases caused by Piscirickettsia salmonis.

[0012] Patent WO2017039454 describes a fish feed comprising conventional feed ingredients and glucosinolates. The feed can be used for the prevention and / or treatment of parasitic infections and / or for interrupting the process of a parasite identifying a fish (host). This document does not mention the use of silibinin as a feed supplement, nor does it mention *P. salmonis* or any bacteria; it refers to parasites, which are different organisms.

[0013] US patent 10967035B1 discloses a feed additive suitable for use in commercial aquaculture, made from whole dried seaweed powder supplemented with 2.5 percent (w / w) andrographolide. This additive increases the feed conversion ratio (the ratio of weight gain to weight of feed used) and the growth rate, while reducing vulnerability to waterborne marine pathogens such as Piscirickettsia salmonis and white spot syndrome virus. The patent does not mention the use of silibinin in the feed supplement. US patent 10967035B1 discloses an additive for inclusion in fish feed that protects fish from infectious diseases.

[0014] The current state of knowledge regarding the use of silibinin in the nutritional and pharmaceutical fields for treating various liver diseases is varied. It is also evident that the use of this compound is constantly evolving.

[0015] The non-patent document “EVALUATION OF THE IMMUNOMODULATING EFFECTS OF SILIMARIN EXTRACT (SILYBUM MARIANUM) ON SOME IMMUNE PARAMETERS OF RAINBOW TROUT, ONCORHYNCHUS MYKISS (ACTINOPTERYGII: SALMONIFORMES: SALMONIDAE)”, by Amhali et al., shows that juvenile rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), were kept in fiber tanks. The fish were administered silymarin extract incorporated into their diets (0.0, 0.1, 0.4, and 0.8 g per 1 kg of feed). The trout were fed this silymarin-supplemented diet for 30 days. Hematological parameters such as red blood cell (RBC) count, white blood cell (WBC) count, hematocrit (Hct), hemoglobin (Hb), leukocyte and differential immunological parameters such as peroxidase, lysozyme and complement activities, total protein levels, albumin and globulin were measured on days 7, 15 and 30 of treatment with silimahna.The results indicated that oral administration of silymarin to fish, after 15 and 30 days of experimental periods, could improve hematological and immunological parameters, including lysozyme and complement activities, total protein levels, and globulin, compared to controls. The conclusion was that the results suggest that oral administration of silymarin may be useful for strengthening the immune system in rainbow trout. This study does not mention salmon, nor does it demonstrate that silymarin is useful in combating infectious diseases caused by Piscirickettsia salmonis.

[0016] Even with all the available information, it is not directly observed that Silibina is used to treat infectious diseases within a food supplement for salmonids.

[0017] Therefore, there is a need for a compound that allows for the care of salmonid species, as well as the protection of the environment. For this reason, a feed supplement for salmonids was developed to treat infections and reduce the use of antibiotics in the salmon farming industry.

[0018] Summary of the invention

[0019] The objective of the present invention is the preparation of a food additive based on Silibinin and / or Quercetin, to combat infectious diseases caused by marine bacterial pathogens.

[0020] The results showed that silibinin and quercetin are able to decrease the intracellular replication of P. salmonis isolates like EM-90 (12201) and like LF-89 (CGR02) when co-incubated with the bacteria in SHK-1 cells. Furthermore, silibinin was found to protect Salmona salar against a challenge with an EM90-like P. salmonis isolate.

[0021] Brief description of the figures

[0022] Figure 1. Viability of SHK-1 cells when incubated for 1 and 15 days with different concentrations of silibinin and quercetin. Experiments were performed in triplicate.

[0023] Figure 2. Viability of RT-GUT cells after incubation for 24 hours with different concentrations of silibinin and quercetin. Experiments were performed in triplicate. Statistical analysis was performed using a one-way non-parametric Mann-Whitney t-test (p-value < 0.05, ns = not significant), comparing the different treatments with control cells (Ctrl).

[0024] Figure 3. Effect of co-incubation of quercetin and silybin on the replication of P. salmonis in the SHK-1 cell line. Three different concentrations of silybin were used: S / 4 (17 pg / mL), S / 2 (34 pg / mL), and S (68 pg / mL), and three different concentrations of quercetin: Q / 4 (7 pg / mL), Q / 2 (14 pg / mL), and Q (27 pg / mL). Infection was performed with two P. salmonis isolates at MOIs of 50 and 200. Intracellular replication of P. salmonis was determined by quantifying the glyA gene. Experiments were performed in triplicate. Stars indicate statistically significant differences between the control (C+) and the treatments. Statistical analysis was performed using a one-way Mann-Whitney U t-test. Figure 4. Effect of co-incubation, pre-incubation and post-incubation of Quercetin and Silibinin on the replication of P. salmonis in the SHK-1 cell line.Three different concentrations of silibinin were used: S / 4 (17 pg / mL), S / 2 (34 pg / mL), and S (68 pg / mL). Three different concentrations of quercetin were also used: Q / 4 (7 pg / mL), Q / 2 (14 pg / mL), and Q (27 pg / mL). Infection was performed using two isolates of P. salmonis at an MOI of 50 and a MOI of 200. Intracellular replication of P. salmonis was determined by quantifying the glyA gene. Experiments were performed in triplicate. Stars indicate statistically significant differences between the control (C+) and the treatments. Statistical analysis was performed using a one-way Mann-Whitney U t-test.

[0025] Figure 5. Effect of co-incubation of quercetin and silybin on the replication of P. salmonis in the SHK-1 and RT-GUT cell lines. Three different concentrations of silybin were used: S / 4 (17 pg / mL), S / 2 (34 pg / mL), and S (68 pg / mL), and three different concentrations of quercetin: Q / 4 (7 pg / mL), Q / 2 (14 pg / mL), and Q (27 pg / mL). Infection was performed with an EM90-like isolate of P. salmonis in a MOI of 50. Intracellular replication of P. salmonis was determined by quantifying the glyA gene. Experiments were performed in triplicate. Stars indicate statistically significant differences between the control (C+) and the treatments. Statistical analysis was performed using a one-way Mann-Whitney U t-test.

[0026] Figure 6. Evaluation of the immunostimulatory effect of incubation with 68 pg / mL of silibinin and with 27 pg / mL of quercetin on SHK-1 cells. A) Incubation for 2 hours. B) Incubation for 24 hours. C (untreated cells), Q (cells incubated with quercetin), S (cells incubated with silibinin). Experiments were performed in triplicate. Statistical analysis was performed using a one-way Mann-Whitney U t-test (p-value < 0.05, ns = not significant), comparing the control (C) with the respective treatments.

[0027] Figure 7. Viability of P. salmonis post-incubation with silibinin and quercetin for 24 hours. Three different concentrations of silibinin were used: S / 4 (17 pg / mL), S / 2 (34 pg / mL), and S (68 pg / mL), and three different concentrations of quercetin: Q / 4 (7 pg / mL), Q / 2 (14 pg / mL), and Q (27 pg / mL). Infection was performed using an EM90-like isolate with an MOI of 50. Experiments were performed in triplicate. Statistical analysis was performed using a one-way Mann-Whitney U t-test (p-value < 0.05, ns = not significant).

[0028] Figure 8. Phagocytosis assay using FluoSpheres carboxylate-modified cells in SHK-1 cells incubated with quercetin and silibinin. The cells were co-incubated for 24 hours with the compounds and microspheres and pre-incubated with the compounds for 24 hours before incubation with the microspheres. Experiments were performed in triplicate. Statistical analysis was performed using a one-way Mann-Whitney non-parametric t-test (p-value < 0.05, ns = not significant).

[0029] Figure 9. Evaluation of the expression levels of P. salmonis virulence factor genes after 24 hours of co-incubation with quercetin (Q) and silibinin (S) during infection in SHK-1 cells. Evaluation was performed on bacteria collected from the supernatant and those collected from the pellet. Experiments were performed in triplicate. Statistical analysis was performed using a one-way Mann-Whitney U t-test (p-value < 0.05, ns = not significant), comparing treatments with the control (Ctrl).

[0030] Figure 10. Evaluation of the effect of administering 27 pg / g of fish of Quercetin (Q) and 68 pg / g of fish of Silibinin (S) for 30 days on the growth of S. salar. Statistical analysis was performed using a one-way non-parametric Mann-Whitney t-test (p value < 0.05, ns = not significant), comparing the treatments with the control (Ctrl).

[0031] Figure 11. Evaluation of the immunostimulatory effect of administering 27 pg / g of fish of quercetin (Q) and 68 pg / g of fish of silybin (S) for 10 days on Atlantic salmon. The effect was evaluated locally, by measuring the immune response in the intestine, and systemically, by measuring the immune response in the kidney. Stars indicate statistically significant differences between the control (Ctrl) and the treatments. Statistical analysis was performed using a one-way Mann-Whitney U non-parametric t-test.

[0032] Figure 12. Evaluation of the immunostimulatory effect of administering 27 pg / g of fish quercetin (Q) and 68 pg / g of fish silybin (S) for 20 days to Atlantic salmon. The effect was evaluated locally by measuring the immune response in the intestine and systemically by measuring the immune response in the kidneys. Stars indicate statistically significant differences between the control (Ctrl) and the treatments. Statistical analysis was performed using a one-way, non-parametric Mann-Whitney t-test. Figure 13. Evaluation of the immunostimulatory effect of administering 27 pg / g of fish quercetin (Q) and 68 pg / g of fish silybin (S) for 30 days to Atlantic salmon. The effect was evaluated locally by measuring the immune response in the intestine and systemically by measuring the immune response in the kidneys. Stars signify statistically significant differences between the control (Ctrl) and the treatments.Statistical analysis was performed using a one-way non-parametric Mann-Whitney t-test.

[0033] Figure 14. Evaluation of the levels of immune response marker transcripts in the kidney of S. salar. The fish were infected with P. salmonis and treated with 27 pg / g of quercetin and 68 pg / g of silibinin. Statistical analysis was performed using a one-way non-parametric Mann-Whitney t-test, comparing the control with the treatments.

[0034] Figure 15. Evaluation of the levels of immune response marker transcripts in the intestine of S. salar. The fish were infected with P. salmonis and treated with 27 pg / g of quercetin and 68 pg / g of silybin. Statistical analysis was performed using a one-way non-parametric Mann-Whitney t-test, comparing the control with the treatments.

[0035] Figure 16. Evaluation of transcript levels of P. salmonis virulence factor genes detected in S. salar kidney. Fish were infected with P. salmonis and treated with 27 pg / g of quercetin (Q) and 68 pg / g of silybin (S). Only genes that showed a statistically significant difference are shown. Statistical analysis was performed using a one-way Mann-Whitney non-parametric t-test, comparing the control (Ctrl) with the treatments.

[0036] Figure 17. Survival percentage of Atlantic salmon challenged with P. salmonis like EM-90 and fed 68 pg / g of Silibina fish. Statistical analysis was performed using the Log-Rank test (Mantel-Cox), p-value < 0.05

[0037] Figure 18. Bacterial loads of dead and surviving fish challenged with P. salmonis fed 68 pg / g of Silibina fish. The presence of P. salmonis was assessed in the intestine and anterior kidney by detecting the 16S rRNA gene.

[0038] Detailed description of the invention

[0039] In this invention, a food supplement for salmonids was developed to treat the infection caused by Piscirickettsia salmonis, and to reduce the use of antibiotics in the salmon industry.

[0040] The results of this technology showed that Silibinin is able to decrease the intracellular replication of the (12201) like EM-90 and (CGR02) like LF-89 isolates of P. salmonis when co-incubated with the bacteria, in a dose-dependent manner. This treatment could be an alternative to the use of antibiotics and can be used palliatively at the time of infection.

[0041] The present invention seeks to protect the use of Silibin as a food supplement for salmonids.

[0042] Silybin is a flavonoid extracted from Silybum marianum, a species of the genus Silybum, and chemically synthesized for commercial use. This molecule can be mixed into commercial salmonid feed through mechanical oiling or incorporated during the feed extrusion process. The inclusion of appropriate concentrations of silybin in salmonid feed can protect against bacterial infections.

[0043] Sili lina does not affect the viability of bacteria as antibiotics do by generating resistance; its mechanism is on the pathogenicity of the bacteria and does not have a stimulating effect on salmonids, therefore its application is palliative and not a preventive treatment.

[0044] In one embodiment, there is a food supplement for salmonids, made from Silibin and / or Quercetin, which serves to combat infectious diseases caused by Piscirickettsia salmonis, in which Silibin or quercetin is mixed into the commercial salmonid feed by mechanical oiling, or incorporated in the feed extrusion process.

[0045] The food additives proposed in this application are Silibinin and Quercetin, which are powder compounds; therefore, they can be included within the extrudate in the pellet creation process, or adhered by mechanical action with an oily solution (commercial oil, fish oil, etc.).

[0046] It is possible to use Quercetin as a replacement for Silibinin, since they are 2 molecules of similar structure that can be used to combat infectious diseases caused by marine bacterial pathogens in fish.

[0047] More specifically, a dietary supplement was developed to treat infection caused by Piscirickettsia salmonis and to reduce antibiotic use in the salmon farming industry. The molecular structures are as follows: Silibilina

[0048] In a preferred embodiment, a food additive for combating infectious diseases caused by marine bacterial pathogens is described, comprising: Silibin in the range of 2-100 pg / mL and / or Quercetin in the range of 2-50 pg / mL in powder form, extruded, or as part of a pellet compound if attached thereto.

[0049] Extruded products typically contain proteins, lipids, ash, and starch, or combinations thereof, in varying percentages. Some components may include fishmeal, soy protein, wheat, vegetable oil, soy flour, various amino acids (histidine, threonine, methionine), antioxidants, and some may contain probiotics, or combinations thereof.

[0050] The advantage of this preparation is that it can be added directly to the food to prepare the additive. In another preferred embodiment, a process for preparing a food additive product is described, comprising:

[0051] Apply Silibin in a range of 2-100 pg / mL or

[0052] Quercine in a range of 2-50 pg / mL within the extrudate for pellet creation, or adhere by mechanical action with oily solution.

[0053] In another preferred embodiment, the aforementioned process is described, where the oil solution can be commercial oil, fish oil, etc., or combinations thereof. Yet another preferred embodiment describes the use of the aforementioned additive, which serves to produce a feed that is useful in combating infectious diseases in fish.

[0054] In another preferred embodiment, the described use is provided where the infectious disease in fish is caused by marine pathogens. In another preferred embodiment, the use is provided where the marine pathogens can be selected from the group consisting of Piscirickettsia salmonis, F. pyschrophilum, and Tencibaculum dicenti.

[0055] The dietary supplement of the present invention is composed of the flavonoids silybin and / or quercetin. In vitro, the antibacterial activity of silybin and quercetin was determined against three isolates of P. salmonis in cell-free liquid medium. Silybin exhibited a minimum inhibitory concentration (MIC) of 128–256 pg / mL and a median maximum inhibitory concentration (IC50) of 68–98 pg / mL, while quercetin had an MIC of 64 pg / mL and an IC50 between 19 and 32 pg / mL. The antibacterial effect was also evaluated on an isolate of F. psychrophilum (Silibinin; MIC 16 pg / mL, IC50 6.4 pg / mL and Quercetin 16 pg / mL, IC50 9.7 pg / mL) and an isolate of T. dicentrachi (Silibinin; MIC 64 pg / mL, IC50 35.2 pg / mL and Quercetin; MIC 32 pg / mL, IC50 18.9 pg / mL) (Table 1). Table 1. Antibacterial activity of Silibinin and Quercetin on the growth of the marine pathogens P. salmonis, F. psychrophilum and T. dicentrachi.

[0056] Furthermore, the cytotoxicity of these compounds was evaluated during 1 and 15 days of incubation in SHK-1 cells, obtaining a cell viability of 92% and 72%, respectively, with the highest concentration tested of Silibinin, while with Quercetin, 57% and 71% were obtained, respectively (Figure 1).

[0057] Experiments conducted under in vitro conditions showed that the compounds have an antibacterial effect against P. salmonis, F. psychrophilum and T. dicentrachi.

[0058] In cell culture experiments, the compound, at sub-IC50 concentrations, decreased the replication of P. salmonis in SHK-1 cells. Furthermore, the compounds stimulated the Th1 cellular immune response, which is essential for combating P. salmonis infection. In vivo experiments showed that the compounds did not affect the growth of Atlantic salmon. However, no stimulation of the immune system was observed in the fish.

[0059] In the challenge experiment of the present invention, an increase in the survival rate of fish treated with silibin compared to control fish was observed when challenged with P. salmonis. These results demonstrate that silibin is non-toxic and can be used in salmonids to provide protection against P. salmonis. Application examples

[0060] Definitions:

[0061] D600 is an abbreviation that indicates the optical density of a sample measured at a wavelength of 600 nm over a 1 cm light path. It is a method commonly used in microbiology to estimate the concentration of bacteria or other cells in a liquid, as the 600 nm wavelength does little to harm or hinder their growth.

[0062] MIC Minimum inhibitory concentration, in microbiology, is the lowest concentration of an antimicrobial that inhibits the growth of a microorganism after incubation.

[0063] IC50 Median Inhibitory Concentration is a measure of the effectiveness of a compound in inhibiting a specific biological activity, such as cell proliferation, enzyme activity, or the viability of a microorganism.

[0064] Example 1: In vitro antibacterial effect of Quercetin and Silibin on Piscirickettsia salmonis, Flavobacterium psychrophilum and Tenacibaculum dicentrarchi.

[0065] To determine the MIC and ICso of quercetin and silybin in P. salmonis, the microdilution method (Weinstein, 2020) was used with some modifications. For this experiment, four P. salmonis isolates (CGR02, 12201, 8149, and 727) were used, which were cultured in cell-free medium (Austral-SRS medium) (Yañez et al., 2012). The bacteria were incubated on Austral-SRS agar plates for 5 days at 18 °C. Subsequently, the bacteria were collected and inoculated into 5 mL of Austral-SRS medium and incubated for 3 days at 18 °C with shaking at 180 rpm. The bacteria were adjusted to OD600 = 0.1 and inoculated again into 10% Austral-SRS medium in a final volume of 5 mL and incubated for 3 days at 18 °C with shaking at 180 rpm.

[0066] Subsequently, P. salmonis isolates at an OD600 = 0.1 were inoculated into a 10% cell suspension in AUSTRAL-SRS medium in 96-well plates (SPL), and treated with the compounds at serial concentrations between 128 pg / mL and 2 pg / mL. The plates were incubated for 96 h at 16 °C with shaking at 180 rpm.

[0067] To evaluate the effect on F. psychrophilum, TYES medium at pH 7.2 was used, inoculating 1% into 10 mL of medium. The bacteria were incubated with agitation for 96 hours at 16°C. Subsequently, the OD600 was adjusted to 0.4, and 1% was inoculated into TYES medium at pH 7.2 in 96-well plates (SPL). These plates were then treated with the compounds at serial concentrations between 128 pg / mL and 2 pg / mL. The plates were incubated for 96 hours at 16°C with agitation at 180 rpm.

[0068] To evaluate the effect on Tenacibaculum dicentrarchi, marine medium was used, inoculated at 10% in a final volume of 5 mL. The bacteria were incubated for 48 h with shaking at 180 rpm. Subsequently, the OD600 was adjusted to 0.1, and the 10% inoculation was applied to marine medium in 96-well plates (SPL). These plates were then treated with the compounds at concentrations ranging from 128 pg / mL to 2 pg / mL. The plates were incubated for 96 h at 16 °C with shaking at 180 rpm.

[0069] To calculate the MIC and ICso of both compounds in the tested bacteria, the OD600 nm of each well was measured using a Nanoquant Infinite M200 Pro (TECAN, Gródig, Austria). The data were analyzed using GraphPad Prism 8.0 software. Concentrations were log(10) transformed, the OD600 nm was normalized as a percentage, and a nonlinear regression was performed to calculate the MIC and ICso.

[0070] The results obtained from the calculation of the MIC and ICso of both compounds in the tested bacteria are shown in Table 1.

[0071] Example 2: Cytotoxic effect of Quercetin and Silibin on the SHK-1 and RT-GUT cell lines.

[0072] To evaluate the toxic effect of the compounds on the SHK-1 and RT-GUT cell lines, both cells were cultured in Leibovitz's L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco). 5Cells were seeded in 24-well plates (SPL) and incubated for 24 hours at 16 °C. The medium was then removed and the cells were washed with 1X PBS (Cytiva, Hyclone). Leibovitzs L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Cytiva, Hyclone) and 40 pM p-mercaptoethanol (Life technologies, Gibco), was added to the cells and they were incubated with 3 sub-inhibitory concentrations of Silibinin (IC50 = 68 pg / mL, IC50 / 2 = 34 pg / mL, IC50 / 4 = 17 pg / mL) and Quercetin (IC50 = 27 pg / mL, IC50 / 2 = 14 pg / mL, IC50 / 4 = 7 pg / mL). The cells were kept for 24 hours at 16 °C. Subsequently, the supernatant from the wells was collected and the cells adhering to the wells were detached using 100 pL of Triple Express Enzyme (ThermoFisher Scientific) for 5 minutes. The cells were collected and centrifuged at 1000 x g for 5 minutes at 4 °C.The supernatant was discarded, and the cell pellet was washed with 300 pL IF buffer (1X PBS, 2% fetal bovine serum) and centrifuged again at 1000 x g for 5 minutes at 4 °C. The supernatant was discarded, and the pellet was resuspended in 300 pL IF buffer (1X PBS, 2% fetal bovine serum). Cell viability was determined by adding 1 pL of propidium iodide (IP, 1 mg / mL), and cells were analyzed by flow cytometry using a FACSCanto II Cytometer (BD Biosciences). Live cells were determined to be IP-negative.

[0073] The results in SHK-1 cells showed that incubation with 3 sub-inhibitory concentrations of Silibinin (IC50 = 68 pg / mL, IC50 / 2 = 34 pg / mL, IC50 / 4 = 17 pg / mL) and Quercetin (IC50 = 27 pg / mL, IC50 / 2 = 14 pg / mL, IC50 / 4 = 7 pg / mL) for 24 hours, showed no effect on cell viability (Figure 1).

[0074] Figure 2 shows the viability of RT-GUT cells incubated for 24 hours with different concentrations of silibinin and quercetin. Experiments were performed in triplicate. Statistical analysis was performed using a one-way Mann-Whitney U t-test (p-value < 0.05, ns = not significant), comparing the different treatments with control cells (Ctrl). The results for RT-GUT cells show that none of the quercetin concentrations used (Q / 4, Q / 2, Q) affected cell viability compared to control cells (Ctrl). However, incubation with 34 pg / mL (S / 2) and 68 pg / mL (S) decreased cell viability by 5% to 7% compared to the control (Ctrl) (Figure 2).

[0075] Example 3: Effect of Quercetin and Silibin on the intracellular replication of P. salmonis in SHK-1 cells

[0076] In the search for a treatment that reduces the probability of resistance development, the ability of these compounds to affect the internalization of P. salmonis in SHK-1 cell culture (macrophage-like cells, derived from the anterior kidney of Atlantic salmon) and its replication inside the cell was evaluated, considering that this step is fundamental for the bacteria and its virulence.

[0077] For this experiment, three sub-inhibitory concentrations of silibinin (IC50 = 68 pg / mL, IC50 / 2 = 34 pg / mL, IC50 / 4 = 17 pg / mL) and quercetin (IC50 = 27 pg / mL, IC50 / 2 = 14 pg / mL, IC50 / 4 = 7 pg / mL) were used. SHK-1 cells were cultured in Leibovitz's L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco). 5Cells were seeded in 24-well plates (SPL) and incubated for 24 hours at 16 °C. Subsequently, the cells were co-incubated with the compounds and P. salmonis MOI:50 for 24 hours with Leibovitzs L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning) and 40 pM p-mercaptoethanol (Life technologies, Gibco). Subsequently, the cells were washed with 1X PBS and incubated for 2 hours with 50 pg / mL gentamicin, washed again with 1X PBS and incubated for 7 days with fresh Leibovitzs L-15 medium in the case of the EM-90-like isolate and for 14 days in the case of the LF-89 isolate. The same experiment was performed by infecting SHK-1 cells with MOI:200.After the infection time had elapsed, the supernatant was collected, while the pellet was incubated with 100 pL of TrypLE Express (Gibco, invitrogen) for 3 minutes, then 500 pL of Leibovitzs L-15 medium (Cytiva, Hyclone) was added, and it was collected for subsequent DNA extraction.

[0078] DNA extraction was performed using the Wizard Genomic DNA Purification Kit (Promega) following the manufacturer's instructions. The DNA was diluted to 50 ng / pL and used for absolute quantification of the bacterial load by detecting the glyA gene of P. salmonis. Quantification was performed using real-time PCR with a PikoReal 96 system (Thermoscientific). The reaction mixture consisted of 5 pL of SsoAdvanced Universal™ SYBR® Green Supermix (Bio-Rad), 0.5 pL of each primer (10 µM), 1 pL of DNA (50 ng), and 3 pL of ultrapure water (Invitrogen) to make a total of 10 pL. The thermal profile used was 1 cycle at 95 °C for 2 min, 35 cycles at 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. To calculate the number of copies of the gene, a previously prepared calibration curve was used.

[0079] The results obtained showed that silibinin is able to decrease the intracellular replication of the P. salmonis like EM-90 and like LF-89 isolates when co-incubated with the bacteria, in a dose-dependent manner, in SHK-1 cells. While quercetin is able to decrease replication of both P. salmonis isolates when co-incubated with the bacteria, but not in a dose-dependent manner, in SHK-1 cells.

[0080] Co-incubation of Silibinin with P. salmonis, isolate EM-90 in an MOI:50, in SHK-1 cells, at a concentration of 17 pg / mL (S / 4), increased bacterial replication by 1.5-fold; the concentration of 34 pg / mL (S / 2) decreased bacterial replication by approximately 19-fold; while the highest concentration tested, 68 pg / mL (S), decreased bacterial replication by approximately 31-fold. Co-incubation of Silibinin with the same isolate, but in an MOL200, showed no significant changes at the lowest concentration tested, while at 34 pg / mL (S / 2) and 68 pg / mL (S), it decreased replication by 2.5-fold and 7.5-fold, respectively (Figure 3).

[0081] Co-incubation of quercetin with P. salmonis isolate EM-90 at an MOI of 50 in SHK-1 cells at a concentration of 7 pg / mL (Q / 4) decreased bacterial replication by 163-fold, at 14 pg / mL (Q / 2) by approximately 83-fold, while the highest concentration tested, 27 pg / mL (Q), decreased bacterial replication by approximately 28-fold. Co-incubation of quercetin with the same isolate at an MOI of 200 at the lowest concentration of 7 pg / mL (Q / 4) decreased replication by approximately 46-fold, while at 14 pg / mL (Q / 2) and 27 pg / mL (Q) it decreased it by 36-fold and 17-fold, respectively (Figure 3). On the other hand, co-incubation of silibinin with P.salmonis, but with an LF-89 isolate in an MOI:50, in SHK-1 cells, at a concentration of 17 pg / mL (S / 4), decreased bacterial replication by 5 times, the concentration of 34 pg / mL (S / 2), decreased bacterial replication by approximately 14 times, while the highest concentration tested, 68 pg / mL (S), decreased bacterial replication by 39 times.

[0082] Co-incubation with silibinin using the same isolate, but in an MOI:200, showed no significant changes at the lowest concentration tested, while at 34 pg / mL (S / 2) and 68 pg / mL (S), it decreased by 6.5 and 33 times, respectively (Figure 3). Finally, co-incubation with quercetin using P. salmonis isolate LF-89 in an MOL50, in SHK-1 cells, at a concentration of 7 pg / mL (Q / 4), decreased bacterial replication by approximately 23 times, while at 14 pg / mL (Q / 2) and 27 pg / mL (Q), it decreased by approximately 17 and 20 times, respectively. Co-incubation of Quercetin with the same isolate, but at an MOI:200, at the lowest concentration 7 pg / mL (Q / 4), decreased replication by approximately 41 times, while with 14 pg / mL (Q / 2) and 27 pg / mL (Q), it decreased by approximately 4 and 5 times, respectively (Figure 3).

[0083] To evaluate whether the effect of silibinin and quercetin on decreasing intracellular replication of P. salmonis in SHK-1 cells is cell- or bacterio-mediated, the effects of incubating SHK-1 cells with the test compounds were compared before infection with P. salmonis (Pre), after infection with P. salmonis (Post), and concurrently with infection with P. salmonis (Co). An EM-90 isolate at MOI:50 and MOI:200 was used for this experiment. In MOI:50, Silibin at the lowest concentration tested (17 pg / mL, S / 4) in co-incubation (co) increased the replication of the P. salmonis isolate by 1.5 times, pre-incubation (pre) showed no significant differences, while post-incubation (post) decreased the replication of the bacteria by approximately 2 times.

[0084] The second tested concentration of Silibinin (34 pg / mL, S / 2), in co-incubation (co), decreased bacterial replication by 19 times, pre-incubation (pre) showed no difference, and post-incubation (post) decreased by

[0085] 4.5 times. The highest concentration of Silibin (68 pg / mL, S), in co-incubation (co) decreased replication by 31 times, pre-incubation (pre) showed no differences, while post-incubation (post) decreased by 7 times (Figure 4).

[0086] Increasing the bacterial dose to an MOI of 200 accentuated the differences between the three incubation protocols. At a concentration of 17 pg / mL (S / 4), none of the incubation protocols showed any changes. At 34 pg / mL (S / 2) and 68 pg / mL (S), only co-incubation (co) resulted in a decrease in

[0087] 2.5 and 7.5 times, respectively. The results obtained with Silibinin show that the greatest effect in decreasing the intracellular replication of P. salmonis is through co-incubation, with a lesser effect when post-incubated in the cells, and no effect when pre-incubated (Figure 4). In the case of Quercetin, when used against a P. salmonis infection at an MOI:50, when co-incubated (co) with 7 pg / mL (Q / 4), it decreases the intracellular replication of P. salmonis by 163 times, pre-incubation (pre) shows no effect, while post-incubation (post) decreases replication by approximately 18 times. With 14 pg / mL (Q / 2), co-incubation (co) decreases the replication of P. salmonis by 83 times, again pre-incubation (pre) has no effect, while post-incubation (post) also decreases the replication of P. salmonis, but only by 23 times.At 27 pg / mL (Q), co-incubation reduces intracellular replication by approximately 28 times, pre-incubation (pre) did not produce changes, while post-incubation decreased by 38 times (Figure 4).

[0088] Increasing the bacterial dose to an MOI of 200 yielded similar results, but with less intensity. At 7 pg / mL (Q / 4), intracellular replication of P. salmonis decreased approximately 46-fold. Pre-incubation (pre) showed no effect, while post-incubation (post) reduced replication approximately 2-fold.

[0089] At 14 pg / mL (Q / 2), co-incubation (co) reduces P. salmonis replication by approximately 36-fold, while pre-incubation (pre) and post-incubation (post) have no effect on P. salmonis replication. Finally, at 27 pg / mL (Q), co-incubation reduces intracellular P. salmonis replication by 17-fold, while pre-incubation (pre) and post-incubation (post) reduce replication by only 3 and 2 times, respectively. The results obtained with quercetin show similar results to those observed with silibinin, with the greatest effect on reducing intracellular P. salmonis replication occurring via co-incubation, a smaller effect after post-incubation, and no effect after pre-incubation (Figure 4).

[0090] Example 4: Effect of Quercetin and Silibin on intracellular replication of P. salmonis in RT-GUT cells.

[0091] To determine whether the effect of Silibinin and Quercetin is cell type dependent, experiments were performed on RT-GUT cells, derived from rainbow trout intestinal epithelial tissue, a cell line different from SHK-1, macrophage-like cells.

[0092] RT-GUT cells were cultured in Leibovitz's L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco). 1x10 5Cells were seeded in 24-well plates (SPL) and incubated for 24 hours at 16 °C. Subsequently, the cells were co-incubated with the compounds and P. salmonis MOI:50 for 24 hours in Leibovitzs L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco). The cells were then washed with 1X PBS and incubated for 2 hours with 50 pg / mL gentamicin, washed again with 1X PBS, and incubated for 7 days in Leibovitzs L-15 medium. The same experiment was performed infecting SHK-1 cells with MQI:200. The supernatant and pellet from the cell culture were collected as previously described. Absolute quantification was determined by detecting the glyA gene of P. salmonis following the protocol described above.

[0093] The results obtained in RT-GUT showed that co-incubation with 27 pg / mL of quercetin (Q) had a significant effect, increasing bacterial replication approximately twofold. In comparison, in the SHK-1 cell line, bacterial replication decreased by approximately 28 times, while the other concentrations of quercetin had no effect on the RT-GUT cell line. On the other hand, co-incubation with 34 pg / mL of silybin (S / 2) decreased bacterial replication approximately twofold, while the same concentration of silybin in SHK-1 decreased bacterial replication approximately 19 times. The other concentrations of silybin had no effect (Figure 5). These results show that the effect of both compounds is cell-type dependent, being more effective in the SHK-1 cell line than in the RT-GUT cell line.

[0094] Example 5: Effect of Quercetin and Silibin incubation on markers of immune response in SHK-1 cells.

[0095] The effect of incubating the compounds in SHK-1 cells on stimulating the TH1-type cellular response, key for defense against P. salmonis, was evaluated. The experiments were performed by incubating 5 x 10 5Cells were cultured in 6-well plates (SPLs) with Leibovitz's L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco) for 24 hours. Subsequently, the cells were incubated for 2 hours and 24 hours at 16°C with 27 pg / mL quercetin (Q) and 68 pg / mL silibinin (S). The cells were washed with 1X PBS and treated with 200 pL TrypLE Express (Gibco, Invitrogen). The cells were collected and stored for subsequent RNA extraction. TriZol (Invitrogen) was used for RNA extraction, following the manufacturer's instructions. Subsequently, the RT reaction was performed using the All-In-One 5X RT MasterMix (ABM) kit, using 2 pg of RNA, 4 pL of the mastermix, and nuclease-free H2O to a final volume of 20 pL. The thermal profile used was 30 min at 37 °C, 10 min at 60 °C, and 3 min at 95 °C.The quantification of transcription levels of the immunological genes (IL-12, TNF-α, IFN-γ) was performed by real-time PCR using a PikoReal 96 real-time PCR system (Thermoscientific). The reaction mixture consisted of 5 pL of SsoAdvanced Universal™ SYBR® Green Supermix (Bio-Rad), 0.5 pL of each primer (10 µM), 1 pL of cDNA, and 3 pL of ultrapure water (Invitrogen) to make a total of 10 pL. The thermal profile used was 1 cycle at 95 °C for 2 min, 35 cycles at 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. To analyze the change in the transcription level of each gene under study, elongation factor 1α (ef1α) was used to normalize gene expression using method 2'. AACT .

[0096] The results showed that after 2 hours of incubation of SHK-1 cells with 27 pg / mL of quercetin (Q), IFN-γ and IL-12 transcript levels increased approximately 16-fold, and TNF-α transcript levels increased 4- to 8-fold. After 24 hours of incubation, only an increase in IL-12 transcript levels of approximately 2-fold was observed.

[0097] In the case of Silibin, incubation of SHK-1 cells with 68 pg / mL (S) for 2 hours increased IFN-γ transcript levels by approximately 2 times, IL-12 by 4 to 8 times, and TNF-α by 4 times. After 24 hours of incubation, IFN-γ transcript levels increased by 64 times, while TNF-α levels increased by 16 times (Figure 6).

[0098] Example 6: Effect of Quercetin and Silibin incubation on the viability of P. salmonis during infection in SHK-1 cells

[0099] The viability of P. salmonis like EM-90 was determined during co-incubation with the 3 concentrations of Quercetin and Silibinin in the SHK-1 cell line. For this experiment, after the aforementioned 24-hour co-incubation, the supernatant was collected, and the viability of bacteria that did not adhere or internalize was determined by adding 1 pL of propidium iodine (IP, 1 mg / mL) and analyzed by flow cytometry using a FACSCanto II Cytometer (BD biosciences). Live bacteria were determined as IP negative.

[0100] The results obtained show that 17 pg / mL (S / 4) and 68 pg / mL (S) do not significantly affect the viability of P. salmonis, while incubation with 34 pg / mL (S / 2) killed approximately 4% of the bacteria.

[0101] On the other hand, incubation with the three concentrations of quercetin killed between 4% and 7% of the bacteria (Figure 7). These results confirm that the sub-IC50 concentrations used do not significantly affect the viability of

[0102] P. salmonis.

[0103] Example 7: Effect of Quercetin and Silibin incubation on the phagocytic capacity of SHK-1 cells

[0104] Considering that these compounds decrease the replication of P. salmonis in the SHK-1 cell line during co-incubation and do not significantly affect the viability of the bacteria, the effect on the phagocytic capacity of the cells was evaluated, using FluoSpheres carboxylate-modified.

[0105] For this experiment, 1x10 5Cells were seeded in 24-well plates (SPLs) and incubated for 24 hours at 16 °C. Subsequently, the cells were co-incubated with FluoSpheres carboxylate-modified and with three sub-inhibitory concentrations of quercetin and silybin for 24 hours. The cells were then detached using 100 pL of Triple Express Enzyme (ThermoFisher Scientific) for 5 minutes. The cells were collected and centrifuged at 1000 x g for 5 minutes at 4 °C. The supernatant was discarded, and the cell pellet was washed three times with 300 pL of IF buffer (1X PBS, 2% fetal bovine serum) and centrifuged again at 1000 x g for 5 minutes at 4 °C. The supernatant was discarded and the pellet was resuspended in 300 pL IF Buffer (PBS 1X, 2% fetal bovine serum).The cells were analyzed by flow cytometry using FACSCanto II Cytometer (BD biosciences), adding 1 pL of propidium iodine (IP, 1 mg / mL), cell populations negative to IP and positive for FITC were considered. The results obtained from the co-incubation between the 3 concentrations of.

[0106] Quercetin (Q / 4, Q / 2, and Q) and FluoSpheres showed a reduction in the detection of these microspheres inside cells, to only approximately 20% compared to untreated control cells. However, when cells were pre-incubated with the three concentrations of quercetin, the percentage of cells capable of phagocytosis was approximately 75% compared to untreated control cells (Figure 8). Although both treatments reduce phagocytosis in a dose-independent manner, co-incubation with microspheres and quercetin resulted in a significant reduction in phagocytosis compared to pre-incubation with quercetin alone.

[0107] On the other hand, incubation with 17 pg / mL of Silibinin (S / 4) decreased phagocytosis to only 67% compared to control cells, while incubation with 34 pg / mL of Silibinin (S / 2) decreased phagocytosis to 50% compared to control cells. Finally, incubation with 68 pg / mL of Silibinin (S) decreased phagocytosis to 25%. However, when cells were pre-incubated with all three concentrations of Silibinin, the percentage of cells capable of phagocytosis was the same as in control cells (Figure 8).

[0108] These results demonstrate that silibinin decreases the phagocytic capacity of SHK-1 cells in a dose-dependent manner when co-incubated with the microspheres, and that this effect is lost when the compound is pre-incubated in the cells. Example 8: Effect of quercetin and silibinin incubation on the expression levels of virulence factor genes in P. salmonis

[0109] The effect of co-incubation of compounds with P. salmonis like EM-90 during infection in SHK-1 cells was determined, on the expression levels of P. salmonis virulence factors.

[0110] For this experiment, SHK-1 cells were cultured in Leibovitzs L-15 medium (Cytiva, Hyclone), supplemented with 10% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco). 1x10 5 Cells were seeded in 24-well plates (SPL) and incubated for 24 hours at 16 °C. Subsequently, the cells were co-incubated with the compounds and P. salmonis MOI:50 for 24 hours using Leibovitzs L-15 medium (Cytiva, Hyclone), supplemented with 5% fetal bovine serum (Cytiva, Hyclone), 4 mM L-glutamine (Mediatech, Corning), and 40 pM p-mercaptoethanol (Life Technologies, Gibco).

[0111] After 24 hours of co-incubation, the supernatant, corresponding to the bacteria that were unable to adhere to the cells, and the pellet, corresponding to the bacteria that were able to adhere to the cells, were collected. The supernatant and pellet from the cell culture were collected as previously described. RNA extraction, cDNA synthesis, and quantification of the transcription levels of the P. salmonis virulence factors (ompA, ospA, MCE2B, cadF, IptD, filC, pilA, pilB, pilQ, dotB, icmK, icmE) were performed following the protocols mentioned above. To analyze the change in the transcription level of each gene under study, the P. salmonis 16S rRNA gel was used to normalize gene expression using method 2. _AACT

[0112] The results showed that in the case of incubation with 27 pg / mL of Silibinin (S), in bacteria collected from the infection supernatant, the expression levels of ompA decreased by approximately twofold, and icmE by approximately fourfold. On the other hand, the transcript levels of mce2B, IptD, pilA, and pilB increased by approximately twofold. Meanwhile, the expression levels of ospA, cadF, filC, pilQ, dotB, and icmK did not show significant changes when compared to the control (Ctrl), which corresponds to untreated bacteria (Figure 9).

[0113] Bacteria collected from cells after 24 hours of infection (pellet) showed an almost twofold increase in the expression levels of ospA, pilQ, and dotB, while the expression levels of ompA, mce2B, cadF, IptD, filC, pilA, pilB, icmE, and icmK did not show significant changes (Figure 9). In the case of co-incubation with 68 pg / mL of Silibinin (S), bacteria collected from the supernatant showed an almost twofold increase in the expression levels of ompA, cadF, pilA, and pilB. No significant changes were observed in the expression levels of ospA, mce2B, IptD, filC, pilQ, dotB, icmK, and icmE (Figure 9).

[0114] On the other hand, bacteria collected from cells (Pellet) showed an increase in the expression levels of ospA and pilQ, close to twofold. Meanwhile, the expression levels of mce2B decreased close to twofold, and IptD, icmE, and icmK decreased close to fourfold. Conversely, the expression levels of ompA, cadF, filC, pilA, pilB, and dotB did not show significant changes when compared to the control (Figure 9).

[0115] These results show that incubating P. salmonis with both compounds alters the expression of virulence factors of P. salmonis.

[0116] Example 9: Effect of Quercetin and Silibinin administration on the growth of S. salar

[0117] Based on the in vitro results, in vivo experiments were conducted to evaluate the effect of administering these compounds to S. salar. 27 pg / g of Quercetin (Q) and 68 pg / g of Silibinin (S) were administered for 30 days, measuring weight and collecting organs every 10 days.

[0118] For this experiment, 24 fish weighing 30 grams each were used per treatment, divided into two ponds with 12 fish each. The fish were fed 2 mm commercial pellets (Biomar), mechanically mixed with commercial oil in the control group, and mechanically mixed with quercetin or silibinin plus commercial oil in the treatment group. Every 10 days, 4 fish per pond (8 per treatment) were weighed, and their kidneys and intestines were extracted and stored in RNAiAter (Invitrogen) for subsequent analysis of immune response markers.

[0119] In the first 10 days of feeding, a statistically significant increase was observed in the fish fed with Quercetin (Q) and Silibinin (S) when compared to the control (Ctrl), between 1 and 2 grams. At 20 days, only the fish fed with Silibinin (S) showed a statistically significant increase, while at 30 days there was no difference between the treatments (Figure 10).

[0120] Example 10: Effect of Quercetin and Silibinin administration on the immune response of S. salar

[0121] For the evaluation of immune response markers in the intestine and kidney of fish treated with 27 pg / g of Quercetin (Q) and 68 pg / g of Silibinin (S), organs from the fish collected in the previous experiment were used for RNA extraction, using 30 mg of organ and Trizol, following the manufacturer's recommendations. cDNA synthesis and quantification of the transcription levels of the immune response markers (IFN-γ, TNF-α, IL-12, IL-1β, TGF-β, lysozyme, and perforin) were performed following the protocols mentioned above. To analyze the change in the transcription level of each gene under study, the elongation factor (ef1α) was used to normalize gene expression using the 2' method. AACT , known to experts in the field.

[0122] The results showed that, after 10 days of treatment, only a slight decrease of 0.4-fold in perforin transcript levels was observed in the intestine of fish treated with quercetin (Q), while in the kidney, only a slight decrease of 0.64-fold in lysozyme transcripts was also observed (Figure 11). After 20 days of treatment, in fish treated with quercetin (Q), a slight decrease in transcript levels of 0.5-fold in lysozyme, 0.3-fold in TGF-β, and 0.8-fold in IL-1β was observed in the kidney. In the case of silibinin (S), a slight decrease in transcript levels of IFN-γ (approximately 0.6-fold), IL-1β (approximately 0.7-fold), TGF-β (approximately 0.5-fold), and lysozyme (approximately 0.8-fold) was observed in the kidney. In the intestine, there was a slight increase in the levels of TGF-p transcript close to 0.3 times, lysozyme close to 0.5 times and perfonna close to 0.4 times (Figure 12).After 30 days of treatment, only a slight decrease in TGF-β transcript levels of approximately 0.3 times was observed in the intestine of fish treated with quercetin (Q) (Figure 13). These results show that the administration of quercetin and silibinin does not significantly modulate the immune response in the kidney and intestine during the evaluated time periods.

[0123] Example 11: Effect of the administration of Quercetin and Silibinin during a P. salmonis infection on the immune response of S. salar

[0124] Considering the results obtained from the evaluation of the immunostimulatory capacity of the compounds, the effect of the administration of 27 pg / g of fish of Quercetin (Q) and 68 pg / g of fish of Silibinin (S) was evaluated during a challenge with P. salmonis in S. salar, on 5 markers of the immune response in S. salar and on virulence factor genes of P. salmonis.

[0125] For this experiment, 12 fish per treatment, weighing approximately 40 grams each, were used, divided into two tanks with six fish each. The fish were fed for five days with 2 mm commercial pellets (Biomar), mechanically mixed with commercial oil in the control group, and mechanically mixed with quercetin or silybin plus commercial oil in the treatment group. On day six, the fish were challenged with P. salmonis like EM-90 via intraperitoneal injection with 3 x 10 5bacteria / gram of fish. Subsequently, the fish were fed their respective treatments for an additional 6 days. Every 3 days, the intestine and kidney of 3 fish per pond (6 fish per treatment) were collected and stored in RNA paste (Invitrogen) for later analysis. RNA extraction from organs and cDNA synthesis were performed as previously described. Quantification of the transcription levels of immune response markers (IFN-γ, TNF-α, IL-12, IL-1β, TGF-β) was performed following the protocols mentioned above. To analyze the change in the transcription level of each gene under study in the S. salar immune system, the elongation factor (ef1α) was used to normalize gene expression using the 2' method. AACT .

[0126] The results showed that, in the kidney, 3 days post-infection, fish challenged and treated with quercetin (Q) showed no statistically significant differences in immune response markers compared to control fish challenged only with P. salmonis (Ctrl). However, 6 days post-infection, an almost 32-fold increase in TNF-α transcript levels and an almost 16-fold increase in IFN-γ transcript levels were observed (Figure 14). On the other hand, fish challenged and treated with silibinin (S) showed an almost 8-fold increase in TNF-α and IFN-γ transcript levels compared to control fish challenged only with P. salmonis (Ctrl). At 6 days, however, only an almost 4-fold increase in IL-1 levels was observed (Figure 14).

[0127] In the intestine, 3 days post-infection, only a nearly two-fold decrease in IL-12 transcript levels was observed in fish challenged and treated with Silibinin (S), compared to control fish challenged only (Ctrl). At 6 days post-challenge, a four- to eight-fold increase in TNF-α transcript levels was observed in fish challenged and treated with Quercetin (Q) and Silibinin (S) compared to control fish challenged only (Ctrl) (Figure 15). These results show that the administration of Silibinin and Quercetin during infection increases the levels of transcripts of genes related to the immune response, which are key to defense against P. salmonis infection, during the first few days of infection.

[0128] Example 12: Effect of administration of Quercetin and Silibin during a P. salmonis infection on P. salmonis virulence factors.

[0129] In the same experiment mentioned above, P. salmonis virulence factor genes were also measured. Transcription levels of the P. salmonis virulence factors (ompA, ospA, MCE2B, cadF, IptD, filC, pilA, pilB, pilQ, dotB, icmK, icmE) were determined following the protocols described previously. To analyze changes in the transcription levels of the P. salmonis virulence factors, the P. salmonis 16S rRNA gel was used to normalize gene expression using the 2' method. AACT At 3 days post-infection, in fish that were challenged and treated with Quercetin (Q), an increase in the levels of transcripts of 2 P. salmonis virulence factor genes was observed, pilB about 2.6 times and pilA about 2.7 times.

[0130] In fish challenged and treated with silibinin (S), transcript levels of three P. salmonis virulence factor genes increased: ospA (3.6-fold), IptD (3.7-fold), and ompA (3.4-fold) (Figure 16). Six days post-infection saw increased transcript levels of eight P. salmonis virulence factor genes in fish challenged and treated with quercetin (Q): ompA (4.8-fold), pilA (5-fold), pilB (4.8-fold), pilQ (4.9-fold), filC (3.3-fold), icmK (5-fold), cadF (6.3-fold), and mce2B (4.7-fold). Silibinin (S) modulated the transcript levels of two P. salmonis virulence factor genes in fish challenged and treated with quercetin. IptD transcript levels decreased 2.6-fold, while pilA transcript levels increased 2.5-fold (Figure 16). These results show that the administration of silibinin and quercetin to S. salar during a challenge with P. salmonis modulates the immune response in S.salt, and also increase the levels of transcripts of different virulence factors of the bacteria, which could explain the mechanism of action of these compounds.

[0131] Example 13: Evaluation of the protective effect of Silibin in S. sa / ar against infection with P. salmonis Finally, the ability of Silibin to protect Atlantic Salmon against infection with P. salmonis was evaluated.

[0132] For this experiment, 18 fish per 50-gram treatment were used, divided into two aquariums with nine fish each. The fish were fed for five days with 2 mm commercial pellets (Biomar), mechanically mixed with commercial oil in the injection control and infection control groups, while the treated group was mechanically mixed with 68 pg / g of silibinin plus commercial oil. On day six, the fish were challenged with P. salmonis like EM-90 via intraperitoneal injection with 3 x 105 Bacteria per gram of fish were measured in the challenge control group and the treated group, while the injection control group received physiological saline solution. Subsequently, the fish were fed their respective treatments for 30 days, and daily mortality was recorded. Anterior kidney and intestine samples were collected from both dead and surviving fish to determine the bacterial load of P. salmonis. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. The bacterial load was quantified by absolute quantification, detecting the glyA gene of P. salmonis, as previously described.

[0133] The results showed that 50% of the fish fed the special diet survived to the end of the experiment, while only 15% of the fish fed the normal diet survived. Furthermore, a statistically significant difference was observed in the survival curve (p < 0.0001) (Figure 17). The presence of *P. salmonis* was confirmed in the anterior kidney and intestine of both dead and surviving fish. The bacterial load of *P. salmonis* in the dead fish was similar between control fish and fish treated with silibinin (Figure 18).

[0134] Based on these in vivo results, it can be concluded that administering silibinin provides protection in Atlantic salmon against infection with P. salmonis. This treatment appears to be an alternative to the use of antibiotics and can be used palliatively at the time of infection.

[0135] It is also possible to use Quercetin instead of Silibinin, since in vitro results showed similar effects in inhibiting intracellular replication of the bacteria, while in vivo results showed similar results in stimulating the immune response during infection, and in increasing the levels of P. salmonis virulence factor transcripts.

[0136] In view of the foregoing, those skilled in the art will understand that changes can be made to the specific aspects described and still achieve the same or a similar result without departing from the spirit and scope of the invention. Therefore, the specific functional and structural details described herein should not be interpreted as exhaustive. It should be understood that the full description of each reference cited herein is incorporated within the description of this application.

[0137] While this invention has been described in the embodiments indicated above, it might seem obvious that other alternatives, modifications, or variations would yield the same results. However, we have established that the subject matter described in this application is fundamental to the success of the invention described herein. Consequently, the embodiments of the invention are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention as defined in the following claims.

[0138] All patents, patent applications, scientific articles and other public documents that, to the knowledge of the applicants, constitute the state of the art, have been duly cited in this application.

Claims

CLAIMS 1. A food additive for combating infectious diseases caused by marine bacterial pathogens, CHARACTERIZED in that it comprises: - Silibin in a range of 2-100 pg / g, and / or - Quercetin in a range of 2-50 pg / g powder, and - Additives for extrusion formation.

2. The food additive of claim 1, CHARACTERIZED in that the extrudate forming additives are selected from proteins, lipids, ash and starch in different percentages.

3. The food additive of claim 1, CHARACTERIZED in that the additives are selected from fishmeal, soy protein, wheat, vegetable oil, soy flour, various amino acids (histidine, threonine, methionine), antioxidants, some may contain probiotics.

4. A process for manufacturing a food additive, CHARACTERIZED in that it comprises: - Have Silibin in a range of 2-100 pg / g, and / or Quercetin in a range of 5-30 pg / g, - Include it within an extrusion for pellet creation, or - adhere by mechanical action with oily solution.

5. The process of claim 4, CHARACTERIZED in that the oily solution can be commercial oil, fish oil, or combinations thereof.

6. The use of the additive of claim 1, CHARACTERIZED in that it serves to manufacture a feed that is useful in combating infectious diseases in fish.

7. The use of claim 6, CHARACTERIZED in that the infectious disease in fish is caused by marine pathogens.

8. The use of claim 7, CHARACTERIZED in that the marine pathogens are defined within the group of Piscirickettsia salmonis, F. pyschrophilum and tencibaculum.

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